龜山島淺海熱泉系統中烏龜怪方蟹共生體之環境互動與適應機制

Abstract

近年來，淺水熱泉因其複雜的特性，受到越來越多的關注，這些特性受到多種表層海洋因素的影響，如日照、潮汐、水流和季節性變化，與深海熱泉有明顯的差異。台灣東北方宜蘭海域的龜山島，同時擁有淺水熱泉及珊瑚生態系統，相當適合作為研究熱泉環境相互作用的自然實驗室。通過監測水質及聲景特徵，我們觀察到熱泉區的間歇性熱液排放，硫化物 (HS-/S2-)、溶解無機碳 (dissolved inorganic carbon, DIC) 和pH值為區分龜山島周圍棲地的重要指標，尤其在活躍期展現了熱泉活動在淺海環境中時間與空間的動態變化。 烏龜怪方蟹 (Xenograpsus testudinatus) 作為龜山島熱泉系統最優勢其中一種動物，已發展出能存活於熱泉極端環境中的生理機制來維持體內恆定。然而，其共生菌與環境變化之間的相互作用仍未完全了解。利用全長16S rRNA基因定序和Alpha多樣性分析，我們觀察到水中細菌群落與熱泉相關的水質參數有相關性。在綱 (class) 的階層下，Campylobacteria和Gammaproteobacteria的相對豐度有相反的變化趨勢，其中，一種硫還原菌Thioreductor是Campylobacteria中相對豐度最高的菌屬，而Gammaproteobacteria中最豐富的菌屬則是一種硫氧化菌Thiomicrorhabdus，說明環境變動與微生物的物質利用之間可能密切相關。而對怪方蟹X. testudinatus來說，鰓的菌群與水質化學變化的相關性高於殼表的細菌，儘管Campylobacteria中的Sulfurovum在鰓和殼表都是優勢菌屬，但殼表的菌群有更高的多樣性。螢光原位雜交 (fluorescence in situ hybridization, FISH) 支持鰓中Sulfurovum的優勢地位。功能預測分析顯示菌群主要進行硫氧化 (sulfur oxidation) 及脫硝作用 (denitrification)，相關反應可能由Sulfurovum主導。由於在海水中Sulfurovum並不是豐度較高的菌屬，怪方蟹的鰓可能為其及相關硫氧化菌 (sulfur-oxidizing bacteria, SOB) 提供了一個適合定殖 (colonization) 的微棲地 (microhabitat)，進而幫助宿主乃至共生體的硫解毒機制 (sulfide detoxification) 及有機物質的提供。 熱泉的間歇性使得X. testudinatus反覆暴露於一般海水與極端環境之間。為了研究牠們的適應機制，我們將實驗室馴化的X. testudinatus回放到原熱泉棲地，採樣並透過定序技術分析鰓的細菌微生物群 (bacterial microbiota)、轉錄體 (transcriptome)，同時測定其氨基酸及脂肪酸組成。在回放轉移後的0, 1, 2, 4小時 (H) 期間，細菌多樣性在2H時觀察到的屬數量 (observed genus taxa number) 和Chao 1顯著下降，Sulfurovum在1H豐度最高，並伴隨著異營性功能 (heterotrophy) 的減少和氮代謝及硫化物代謝的增加，同時轉錄組顯示宿主組織中GTPase活性和肌動蛋白骨架重構 (actin cytoskeleton remodeling) 相關的路徑被上調，而幾丁質和外骨骼的形成 (chitin and cuticle formation) 受到抑制。在之後的2H和4H，硫化物代謝有逐漸增強的趨勢，與硫化物解毒相關的氨基酸牛磺酸 (taurine) 也顯著減少，而與氮代謝相關的組氨酸 (histidine)、精氨酸 (arginine) 和谷氨酰胺 (glutamine) 則在2H及4H時累積，同時與上述胺基酸相關的代謝酵素基因如谷氨酰胺酶 (glutaminase)、組氨酸氨基水解酶 (histidine ammonia-lyase) 及丙氨酸轉氨酶 (alanine transaminase) 也被上調。抗氧化防禦 (antioxidant defense) 則藉由穀胱甘肽過氧化酶 (glutathione peroxidase) 及麩胱甘肽轉移酶 (glutathione S- transferase) 的旁系同原體 (paralogs) 在回放過程中持續進行，說明抗氧化機制對烏龜怪方蟹適應熱泉環境的重要性。此外，脂肪酸含量雖然並沒有顯著變化，相關性和倍數差異 (fold change) 分析顯示不飽和脂肪酸可能比飽和脂肪酸對環境的反應更加明顯，脂肪酸的累積與消耗可能與細胞結構的維持及抗氧化物的調節相關。 上述結果顯示X. testudinatus熱泉共生體棲息於一個比以往認知更為複雜的動態環境中，我們的研究透過多方面的分析，包含環境變量、細菌群時間性變化以及宿主生理代謝反應，探討不同層面之間的交互作用及相互依賴關係。研究熱泉共生體為我們提供了生命在挑戰性環境中所展現出韌性的相關見解，並且揭示了極端條件下跨域共演化 (inter-domain evolution) 的奧秘。

Shallow-water hydrothermal vents have garnered growing attention due to their complex characteristics, influenced by various epipelagic factors such as insolation, tides, currents, and seasonal changes, in contrast to their deep-sea analogues. Kueishan Island, which harbors both shallow-water hydrothermal and coral ecosystems, provides an exceptional natural laboratory for investigating hydrothermal environmental interactions. We captured sporadic fluid discharges at the vent site by monitoring water quality and soundscape features. Sulfide (HS-/S2-), dissolved inorganic carbon (DIC), and pH levels were identified as key indicators for habitats differentiation around Kueishan Island, especially during active venting periods, which highlights the profound spatial and temporal dynamics of hydrothermal activity in shallow water marine environments. The brachyuran vent crab Xenograpsus testudinatus, a dominant metazoan inhabiting Kueishan Island hydrothermal vent system, has developed robust physiological mechanisms to sustain homeostasis in this extreme environment. However, the interactions between its symbiotic bacteria and environmental fluctuations remain incompletely understood. Through the application of full-length 16S rRNA gene sequencing and alpha diversity analysis, we observed strong correlations between water bacterial community and hydrothermal-associated physicochemical parameters at the vent site. The relative abundances of Campylobacteria and Gammaproteobacteria exhibited inverse temporal patterns, where Thioreductor, a sulfur-reducing bacteria, was the most abundant genus within Campylobacteria, while the sulfur-oxidizing Thiomicrorhabdus predominated among Gammaproteobacteria, indicating a potential link between environmental dynamics and microbial resource availability. In X. testudinatus, gill-associated bacteria showed greater correlations with water quality compared to those present on the carapace surface. While Sulfurovum (Campylobacteria) was predominant in both the gills and carapace surface, the carapace surface demonstrated higher bacterial diversity. Furthermore, fluorescence in situ hybridization (FISH) and functional prediction analysis supported the branchial predominance of Sulfurovum, which performs sulfur oxidation and denitrification processes. Given that Sulfurovum is not abundant in the ambient seawater, the gills may serve as an enclave for sulfur-oxidizing bacteria (SOB) such as Sulfurovum, facilitating sulfide detoxification and organic production to the host. The intermittent nature of hydrothermal venting repeatedly exposes X. testudinatus to transitions between normal seawater and extreme environmental conditions. To investigate their adaptive responses, we reintroduced lab-acclimated X. testudinatus to their native habitat and analyzed their gill tissue for bacterial microbiota, transcriptome, amino acid compositions, and fatty acid profiles. During the period, 0, 1, 2, and 4 hours (H) post-reintroduction, bacterial diversity showed a significant reduction in observed taxa numbers and Chao 1 estimates at the genus level at 2H. Sulfurovum had the highest abundance at 1H, coinciding with elevated nitrogen and sulfide metabolism and reduced heterotrophy-associated functions. Meanwhile, transcriptomic analysis revealed upregulation of GTPase activity and actin cytoskeleton remodeling in the host tissue, while chitin and cuticle formation pathways were suppressed. At subsequent time points (2H or 4H), both sulfide metabolism and stress responses showed progressive enhancement. Taurine, crucial for sulfide detoxification, significantly depleted at 2 and 4H. Additionally, nitrogen metabolism-related amino acids, histidine, arginine, and glutamine, accumulated at the later time points, accompanied by upregulation of related enzyme genes, including glutaminase, histidine ammonia-lyase, and alanine transaminase. During the reintroduction, fatty acid levels did not show significant changes; however, correlation and fold change analyses revealed that unsaturated fatty acids had more pronounced responses to environmental transition compared to saturated fatty acids. These analyses demonstrate that X. testudinatus holobiont exists in a dynamic environment, more complicated than previously acknowledged. Our study provides a multi-faceted examination of environmental variables, bacteria community dynamics, and the crab host’s physiological responses, elucidating their intricate interactions and interdependencies. Exploring hydrothermal-endemic holobionts provides valuable insights into the remarkable resilience of life in a challenging environment, shedding light on the mechanisms of inter-kingdom coevolution under extreme conditions.